Addis Ababa University

Institute of Biotechnology

Design of Immunodiagnostic Assays for Detection of Shiga Toxin Producing

Pathogenic E. coli(STEC) Strains Using Polyclonal Antibodies

A Thesis Submitted to the School of Graduate Studies, Addis Ababa University, in Partial
Fulfillment of the Requirements for the Degree of Master of Science in Biotechnology

By

BiniamMoges

Advisor: TesfayeSisay (PhD, Assoc. Prof)

Institute of Biotechnology, Addis Ababa University

March, 2019

Addis Ababa, Ethiopia

I
 Table of Contents

TABLE OF CONTENTS…...................................................................................................................II

ACKNOWLEDGMENTS....................................................................................................................IV

LIST OF TABLES.................................................................................................................................V

LIST OF FIGURES..............................................................................................................................VI

ABBREVIATIONS.............................................................................................................................VII

ABSTRACT...................................................................................................................................... VIII

1. INTRODUCTION.............................................................................................................................1
2. OBJECTIVES OF THE STUDY......................................................................................................6
2.2............................................................................................................Specific objective of the study
...........................................................................................................................................................6
3. LITERATURE RIEVIEW................................................................................................................7
3.1...................................General Overview of Shiga Toxin-Producing Escherichia coli as a pathogen
...........................................................................................................................................................7
3.1.1. Pathogenicity Mechanism of STEC..........................................................................................10
3.1.2. Host immune response against STEC strain infection.............................................................13
3.1.3. Prevalence(Occurance ) of Shiga Toxin-Producing Escherichia coli (STEC)..........................16
3.2...............................................................................Diagnosis/ Detection Mechanism of STEC Strain
.........................................................................................................................................................19
3.2.1. Culture based methods for detection of shiga toxin-producing E. coli.....................................20
3.2.2. Tissue culture cytotoxicity assays for detection of STEC.........................................................22
3.2.3. Immunological methods for STEC strain Detection.................................................................23
3.2.4. Molecular based detection of shiga toxin-producing E. coli.....................................................26
3.2.5. Other emerging technologies for STEC strain detection.......................................................28
3.3....................................Development of immunodiagnostic kit for STEC Detection Using Antibody
.........................................................................................................................................................28
3.3.1. Antigenic parts of STEC selection, isolation and purification for antibody production ..........28
3.3.2. Antibody (monoclonal and polyclonal) Development..............................................................31

II
4. MATERIALS AND METHODS....................................................................................................37
4.1..................................................................................Bacterial strains isolates and growth conditions
.........................................................................................................................................................37
4.2..............Prepare antigen (Lipopolysaccharide-O antigen) of STEC strain by chloroform-methanol
method.............................................................................................................................................37
4.3........Determine the purity of extracted LPS by (SDS-PAGE) followed by commassie blue staining
.........................................................................................................................................................38
4.4......................................................Expermental Animal Selection and Management (Ethical Based)
.........................................................................................................................................................39
4.5.............................Production and Purification of Mice Polyclonal Antibodies Against STEC Strain
.........................................................................................................................................................39
4.6...............Development of slide agglutination assay for STEC strain detection using LPS of STEC
.........................................................................................................................................................41
4.7....................................Evaluation of the specificity of the newly designed immunodiagnostic assay
.........................................................................................................................................................41
4.8....................................Evaluation of the sensitivity of the newly designed immunodiagnostic assay
.........................................................................................................................................................42
4.9........................................Evaluating the detection capacity of the designed assays in spiked sample
.........................................................................................................................................................42

5. EXPECTED OUT PUT (RESULT).....................................................................................................................43

6. DATA MANAGEMENT AND ANALYSIS.................................................................................43

7. Time Line/Work plane of the study.............................................................................................................44
8. Cost of the project......................................................................................................................................45
9. REFERENCE..................................................................................................................................51
10. Annexes...........................................................................................................................................55

10.1.......................................................................................................................Advisor Name and CV

........................................................................................................................................................55
10.2...................................................................................................................... Authors Name and CV
.......................................................................................................................................................83

III
 ACKNOWLEDGMENTS

First and for most, I would like to address my sincerest thanks to my advisor, Dr. Tesfaye Sisay
(Assoc prof.), Institution of Biotechnology, Addis Ababa University, Ethiopia for his invaluable,
wise and a very accadamical guidance and unreserved interest to help me to understand the
scientific background of the study and friendly approach to follow my research activity and
corrected my thesis proposal until the final submission of this proposal. He also support me by
invite to involve his project which is “Design of Immunodiagnostic Assays for Detection of
Shiga Toxin Producing Pathogenic E. coli (STEC) Strains Using Polyclonal Antibodies”, funded
by Ethiopian Biotechnology Institute.

Finally, I would like to use this opportunity to forward my acknowledgement to my family and
colleagues for their indispensable consultation, support and continuous encouragement for the
successful submission of this research proposal.

IV
LIST OF TABLES

Table 1: Pathogenicity genes in shiga toxin serotypes and their promoter function in cells................11

Table 2: STEC O157:H7 virulence factor combinations from studies in Africa..................................18

Table 3: Different typepes of medium and methodologies used to detect STEC.................................21

Table 4: Detection methods by PCR of STEC-associated target genes..............................................27

V
LIST OF FIGURE

Figure 1: General overview of pathogenic gene acquisition and loss for different pathotypes..............8

Figure 2: Mechanism of action of the shiga toxin.................................................................................13

Figure 3: Innate and adaptive immune response aganst Gram negative bacteria (STEC Strain).........16

Figure 4: STEC isolation from various selective media.......................................................................21

Figure 5: Enzyme Linked Immunosorbent Assay (ELISA) methods with different format ................24

Figure 6: Slide agglutination test result.................................................................................................25

Figuer 7: Structure of a lipopolysaccharide of STEC strain.................................................................30

Figure 8: Antibody development (B-cell proliferate in to plasma and memory B-cell).......................32

Figure 9: Structure of an antibody (immunoglobine) Molecule...........................................................33

Figurer 10: polyclonal and monoclonal antibody production scheme..................................................35

Figure 11: Immunoglobulin diversification and B cell development..................................................35

VI
 ABBREVIATIONS

E.Coli- Eshercia coli

EHEC- Entero-hemorrageic Escherichia coli 

EIEC- Entero-invasive Escherichia coli 

EPEC- Entero-pathogenic Escherichia coli 

EPHI - Ethiopian Public Health Institute

ETEC- Entero-toxigenic Escherichia coli 

Gb3 - Globotriaosylceramide-3

HC - Haemorrhagic colitis

HUS - Hemolytic uremic syndrome

IFNγ - Interferon-gamma

LPS - lipopolysaccharide

mAb – Monoclonal antibody

pAb- Polyclonal antibody

PCR – polymerase chain reaction

VII
sELISA - SandwitchEnzyme-Linked Immunosorbent Assay

STEC – Shigatoxine producing Eshercia coli

Stx - Shiga toxins

TMB - Tetramethylbenzidine-

TLRs - Toll-like receptors

VTEC- Vero-toxigenic Escherichia coli 

WHO - World health organization

ABSTRACT

Shiga toxin-producing Escherichia coli (STEC) are the leading pathogenic strain causes of

severe human diseases ranging from diarrhea to hemolytic uremic syndrome by produced one or
more shiga toxins (stx1, stx2, or their virulence variants), which inhibits protein synthesis of host
cells, thus leading to cell death. According to WHO, (2017) report, diarrheal disease due to
STEC and other pathogenic E. coli strain infection are the most frequent and the second leading
causes of death in the world. Over the years, many immunodiagnostic assays to aid in the
diagnosis of STEC strain have been developed; however, many of the available diagnostic assays
take a long time and require expensive equipment and trained personnel may not be readily
available in many areas. Therefore, this problem with detection methods could be minimized by
designed new approaches that can detect all STEC strains with rapidly, sensitively, specifically
and less cost are necessary. To achieve; this study will be begin with isolation of immunogenic
part (LPS) of STEC and production of polyclonal antibody against LPS of STEC strain by
immunized mice. then a simple, rapid, cost effective, sensitive and specific slide
immunodiagnostic assay kit will be developed by using the generated polyclonal antibody for the
detection of STEC strains, followed by the newly designed immunodiagnostic assay kit
detection Limit (Sensitivity) and specificity on bacterial culture and spiked faeces will be
evaluated, and finally, the detection capacity and efficiency of the designed assays in human

VIII
and animal stools and meat samples will be cheeked and discussed. At the last of this study; this
assay will be useful for solving current problems that facing in the detection of STEC strain with
a high specific and sensitive value and provide a valuable tool for health care and research area
from animal, human and environmental samples.

Keywords: Lipopolysaccharide (LPS), Shiga Toxin Producing E. coli (STEC) ,Pathogene

Slide Agglutination, Polyclonal Antibody (pAb), Immunodiagnostic Assays Kit

IX
 1. INTRODUCTION

Escherichia coli was first discovered in 1885 by Theodor Escherich, a German bacteriologist

and they are a facultative (aerobic and anaerobic growth) gram-negative, rod shaped
flagellated bacterium family of Enterobacteriaceae that can normally live in the intestines of
people and animals. Most E. coli bacteria strains are commensals, harmless and an important
part of a healthy human intestinal tract. Some E. coli strains, however, have developed the
ability to cause disease/ pathogenic, either diarrhea or illness outside of the intestinal tract
due to additional multiple virulence genes such as eae, toxin (stxs), found in chromosomes
and/or plasmids.This pathogenicity of E-coliis developed by getting a foreign gene that
coded virulence factor (s) from pathogenic bacterial strain through horizontal/ computation
gene transfer process from a variety of mobile genetic elements such as plasmids,
bacteriophages, transposons and pathogenicity islands(Nataro and Kaper, 1998). This
genomic plasticity implies ongoing re-assortment of virulence factors that complicates to
categorize the various subgroups into sharply delineated pathotypes (Tania et al, 2016). E.
coli can transfer its DNA materials through bacterial conjugation with other related bacteria
to produce more mutation and add more strains into the existing population. This dynamic
characteristic of pathogenic E. coli leads to new challenges in the diagnosis, treatment, and
prevention of E. coli infections (PubMed Health, 2015).

E. coli bacteria strain can be classified into hundreds of serotypes on the basis of different
serotypes of E.col O157:H7, and non - O157:H7. And also pathogenic E. coli strains can be
categorized based on elements that can elicit an immune response in animals, namely: O
antigen (part of lipopolysaccharide layer encoded by the rfb,wzx and wzzy gene cluster), K
antigen (capsule) and H antigen (flagellin- encoded by fliC gene)( Beutin and Strauch,
2006). Certain strains of E. coli, such as O26:H11 or H-, O45:H28, O91:H21 or H-,
O103:H2, O104:H4, O111: H-, O113:H21, O121:H19, 0128: H11, O145:H28 or H- and,
O157:H7 are the most potential lethal toxin produced strains and leads to diarrhea or illness
(Fratamico et al, 2016). Currently, based on their serological characteristics and virulence
(pathotypes) properties, six pathotypes of E. coli are commonly recognized as diarrheagenic
E. coli strain that causes diarrhea disease as result of entero-toxin production, adherence, or

1
invasion, such as entero-toxigenic E.coli (ETEC), entero-pathogenic E.coli (EPEC), entero-
invasive E.coli (EIEC), entero-hemorrageic E.coli (EHEC), entero-adherent aggregative E.
coli (EAggEC), and vero-toxigenic E. coli (VTEC) (James et al, 2006).

Among these, entero-hemorrageic E.coli (EHEC), and shiga toxin producing pathogenic E.

coli (STEC) strain are the leading pathogenic E. coli that cause of diarrhea in children
especially in the developing world, as well as the most common cause of traveler's diarrhea.
STEC cause serious disease in humans is related to the production of one or more Shiga
toxins (stx1, stx2, or their virulence variants), which inhibits protein synthesis of host cells,
thus leading to cell death. The two Stx1 and Stx2 produced by STEC have similar structure,
A-sub unit which is an enzymatically active N-glycosidase that inhibits protein synthesis by
cleavage of an adenine base from the 28SrRNA component of the eukaryotic ribosomal 60S
subunit, resulting in cell death. The B-subunit recognizes and binds to receptors of the target
cells leading to the internalization of the toxin (stx1 and stx2). From STEC strain, only stx2
producing strain is more toxic than strain that express both Stx1 and Stx2; this due to
competition for the same host cell receptors (Hajra et al, 2007). Their toxins are delivered
through the colonization factor (CF), leading to activation of cyclicadenosine
monophosphate and subsequently leading to active water and electrolyte secretion and
diarrhea. This process leads totissue invasion by the organism and destruction of the mucosa
membrane. Diarrheagenic STEC E.coli strain can be transmitted through contaminated water
or food, or through contact with animals or persons (Jabbar, 2003).

Globally, there are nearly 1.7 billion cases of childhood diarrheal disease every year.
Previous studies showed that diarrheal disease are amongst the most frequent and the second
leading causes of the death of 700,000 under-five children every year in the world. Children
specially in developing countries get exposed to many bacterial enteric pathogens at very
early age and suffer many episodes of diarrhea through contaminated food or drinking-water,
or from person-to-person as a result of poor hygiene that lead to the release of important
components (water and electrolytes - sodium, chloride, potassium and bicarbonate) of the
infected person (WHO, 2017).

2
Acute diarrheal diseases due to pathogenic E. coli strain infection are widespread throughout
the world specially developing countries. Children who are malnourished or have impaired
immunity system as well as people living with HIV and other infectious disease are most at
risk of life-threatening diarrhea disease. Infection is more common when there is a shortage
of adequate sanitation and hygiene and safe water for drinking, cooking and cleaning.
Worldwide, 780 million individuals lack access to improved drinking-water and 2.5 billion
lack improved sanitation. Different studies have shown that, in developing countries like;
Africa and South Asia, the occurrence of acute watery diarrheal (AWD) disease among under
five children is complex important public health problems causing substantial mortality and
affect the interaction between socioeconomic, environmental, and behavioral variables. It
occurs both as a short outbreak and protracted epidemic/pandemic. As a result pathogenic E.
coli (diarrheagenic), strains are the most common agents of moderate-to-severe diarrhea
disease (Mohammed and Tamiru, 2014).

Among the entro-pathogenic strains STEC O157:H7, is one of the most common causes of
foodborne infections that leading cause of illness and death of humans in worldwide
especially in developing countries costing billions of dollars in medical care and social costs.
In developing countries especially in Africa continents, enteric pathogen identification is
frequently time consuming and incomplete, that leads to potential misdiagnoses or
mistreatments (Botkin et al., 2012).

To eliminate or eradicate this global health problem by applying effective treatment and
prevention of E. coli infections, different researches indicate that accurate, reliable, low cost,
highly specific and sensitive diagnosis kit and technique for detecting pathogen such as
STEC is needed. Due to the continuous increase in outbreaks of shiga toxin producing
pathogenic E.coli strain, the existing convectional biochemical markers based diagnosis
strategy for identification of unique sorbitol negative fermentation properties of the
previously existing pathogenic E.coli (O157) strain are not sufficient. Why because
currently, more illnesses due to non-O157E.coli strain serotypes are not covered by the
existing diagnostic strategy. According to report published in 2012, the big six non-O157
STEC strains were revealed to be responsible for 113,000 illnesses annually in the United

3
States, almost double the amount of the illnesses caused by E. coli O157 STEC strain and
some cases of non-O157 illness appear to be as severe as cases associated with
O157. Therefore, these existing detection strategy/ technologies are continuously being
refined /improved to have capable of detecting the most prevalence STEC pathogenic E. coli
strains. Not only this but there is also a great need for improved extraction methods of
sample preparation that are less expensive and compatible with rapid assays for the
possibility to have the near-real-time detection of pathogens(He et.al, 2013).

Over the years, many immunodiagnostic assays (from stool culture to molecular gene level)
have been developed to aid in the diagnosis of pathogenic E. coli including STEC strains
strain and they are available commercially; however, a reliable assay for use in local clinics
still remains elusive. Also, multiple studies showed that many of the commercially available
diagnostic assays have limitation such as can’t detects all subset of STEC pathogenic E. coli
strains, give high percentage of false-positive pathogenic results when other pathogens are
present, take a long time, require expensive sophisticated equipment and trained personnel,
less sensitive and specific and gene level diagnostic kit like PCR are also more costly, labor
intensive and can’t detect the toxin itself, but less time-consuming, highly specific and
sensitive to detect the toxin gene(Jabbar, 2003).
Furthermore a subsequent simple, fast, sensitive, specific and cost-effective antibody-based
neutralization immunodiagnostic assay is required for simultaneous detect and identify
multiple pathogens E. coli strain from non-pathogenic strain by generating specific pAb,
because pAbs usually have higher reactivity than mAbs due to the polyvalency (multiple
epitopes to react with) (hajra et al, 2007) versus the monovalency of a mAb (Payne et al.,
1988).
To address these existing diagnostic problems of the STEC pathogenic E .coli strain, this
research/study will be focused on designing a simple, fast, sensitive, specific and cost-
effective immunediagnostic assay for detection of shiga toxin producing pathogenic E. coli
strain (STEC) using or by producing specific polyclonal antibodies. To achieve; this study
will be begin with generation or isolation of immunogenic part (LPS ) of STEC strain and
development of polyclonal antibody against cell surface (LPS) antigen of STEC strain by
immunized mice. Followed by slide agglutination immunodiagnostic assay kit will be

4
developed by using the generated polyclonal antibody. Finally after the designing of
diagnostic assay, the detection capacity and efficiency (sensitivity and specificity) of the
designed assays for detection of STEC strain on culture and spiked faeces will be evaluated
and discussed.
At the last of this study; this assay will be useful for solving current problems that facing in
the detection of pathogenic E. coli strain with a high specific and sensitive value and provide
a valuable tool for health care and research area from animal, human and environmental
samples.

5
 2. OBJECTIVES OF THE STUDY

 General objective of the study

The general objective of this study will be the generation of a polyclonal antibody specific
for STEC strains and for use in the development or designing of a simple, rapid, cost
effective, sensitive and specific immunodiagnostic assay for the detection of STEC strains.

 Specific objectives of the study

 To produce a polyclonal antibody specific to STEC strains.

 To design immunodiagnostic assays using polyclonal antibodies for detection of STEC

strain.

 To determine the sensitivity and specificity of the designed/developed diagnostic assays.

 To validate the performance of the designed assays in detection of STEC strain from fece
samples spiked with known CFUs of STEC strain isolates.

6
 3. LITERATURE RIEVIEW
 General Overview of Shiga Toxin-Producing Escherichia coli as a Pathogen

Historically, Escherichia coli were first discovered in 1885 by Theodore Escherich a German
bacteriologist as a Gram-negative, catalase positive, nitrate reductase positive, indol positive,
oxidase-negative, rod-shaped flagellated bacterium, facultative (aerobic and anaerobic
growth) family of Enterobacteriaceae. They categorized as among the most studied
organisms and serves as the basis for understanding many fundamental biochemical and
genetic concepts. Over 125 years later, E. coli was acquired as a commensal and harmless
bacterium of the gastrointestinal tract in human and various warm-blooded animals’ species
and is used as the colloquial laboratory workhorse (Koev et al., 2017).

However, some pathogenic E. coli that have the ability to cause disease/ pathogenic, either
diarrhea or illness outside of the intestinal tract are evolved from commensal E coli through
the acquisition of multiple virulence determinants (Donnenberg et al., 2001) such as toxins,
adhesins/ fimbriae, bundle-forming pilus gene (bfp), colonization factors (CFs) and secreted
effector proteins which is locus enterocyte effacement (LEE), the acquisition of mobile
genetic elements such as plasmids, insertion sequences, bacteriophages, transposons and
pathogenicity islands, can present either integrated into the chromosome or through self-
replication within the new host to provide highly diverse new adapted traits/pathogen. The
combined effects of different multiple virulence factors determine the extent of E coli
pathogenesis and the severity of human disease (Castro et al., 2017).
Based on their pathotypes or the mechanism they cause disease, pathogenic E.coli strain
grouped as enteropathogenic E coli (EPEC), atypical enteropathogenic, enterotoxigenic E
coli, diffusely adherent E coli(DAEC), enteroaggregative E coli(EaggEC or AAEC),
enterohemorrhagic E coli (EHEC), enteroinvasive E coli(EIEC), adherent-invasive E coli,
sepsis/ meningitis-causing E coli and uropathogenic E coli (figure 1) (Clements et al.,2012).

7
Figure 1: General overview of pathogenic gene acquisition and loss for different
pathotypes

In addation, E coli strains are further characterized according to antigenic variants including
O-antigen (lipopolysaccharide), H-antigen (flagellar) and K-antigen (capsular) types (Croxen
et al., 2013).
Among these pathogenic E coli shiga toxin-producing E coli (STEC) group being the main
representative regarding foodborne pathogenesis with outbreaks worldewide due to the
presence of 2 distinct toxins (stx1 and stx2) and other virulence factors. Shiga toxin-
producing Escherichia coli (STEC) were first discovered in 1977 and first associated with the
clinical syndrome hemolytic-uremic syndrome (HUS) in 1983. Since, diarrhea-associated
HUS has been recognized as one of the most common causes of acute renal failure in
children in the United States (Proulx et al., 2001).

8
STEC Strains that expressing both Stx1 and Stx2 is less toxic than those expressing only
Stx2. This is due to competition for the same host cell receptors. Previous assays showed that
Stx2 was 100 times more toxic to mice than Stx1. In contrast to Stx1 (stx1a, stx1c, and
stx1d), more subtypes have been identified for Stx2. Currently, there are seven predominant
Stx2 subtypes (stx2a, stx2b, stx2c, stx2d, stx2e, stx2f, and stx2g), all of which have been
associated with human illnesses (He et al., 2013). STEC species are classified into 2
subtypes: O157 and non-O157, with cases involving O157 strains more frequently associated
with more severe diseases, such as the development of diarrhea, haemorrhagic colitis (HC)
and hemolytic uremic syndrome (HUS) (Hughes et al.,2006). Non O157 serogroups are the
most prevalent microorganisms in reported foodborne outbreaks in the United States and in
other countries (Monaghan et al., 2011).
Bothe Stx1 and Stx2 produced by STEC strain is an AB5 toxin with a catalytically active A
subunit and five identical B-subunits (Tozzoli, et al., 2009). This subunit forms an A2/B5
complex giving it greater stability and also responsible for recognizing and binding to the
glycolipid surface receptors (specifically the glycolipid globotriaosylceramide (Gb3) of the
target cells leading to the internalization of the toxin (stx1 and stx2). Amino acid (AA)
sequence variations across Stx types and subtypes can result in differences in toxicity. The
AA variability is due to primarily substitutions that occur in the A2 fragment and the B-
subunit. In contrast, there are fewer AA substitutions across subtypes in the catalytically-
active A1 fragment of the A-subunit. This suggests that differences in the toxicity of Stx1,
Stx2, and their subtypes have less to do with differences in A1 toxicity (James et al., 1998).

STEC O157:H7, is one of the most common causes of foodborne infections that leading
cause of illness and death of humans in worldwide especially in developing countries costing
billions of dollars in medical care and social costs. In developing countries especially in
Africa continents, enteric pathogen identification is frequently time consuming and
incomplete, that leads to potential misdiagnoses or mistreatments. Therefore, a rapid,
sensitive and specific assay designed to identify STEC and other strains in a public health
setting would be advantageous to help ensure that a timely and proper response is
initiated(Croxen et al., 2013). Specific identification of highly pathogenic STEC would also
be critical in the event of food-borne illness outbreaks (Botkin et al., 2012).

9
 The existing strategy for detection of clinical samples relies on biochemical markers are
based on the unique sorbitol negative fermentation and ßD-glucuronidase-positive properties
of the E. coli O157: an H7 strain, which is the most commonly, identified STEC serotype
from patients who have HUS and HC (Ranjbar et al., 2018). However, that numerous non-
O157 STEC serotypes also cause outbreaks and severe more illnesses linked to non-O157
STEC serotypes are uncovered. Different studies were conducted and their result published
in 2012 showes that, the big six non-O157 strains were revealed to be responsible for
113,000 illnesses annually in the United States, almost double the amount of the illnesses
caused by E. coli O157. Which indicated that it is necessary to designed or have new
methods that are capable of detecting all STEC strain (He et al., 2013).

3..1. Pathogenicity Mechanism of STEC

The pathogenicity mechanism of STEC such as E coli O157:H7 and other serotype is a multi
step process involving different complex interactions between STEC bacteria and host cell.
The resistance to acidic environments of the stomach by STEC bacteria plays an important
role in favoring the infection process of the host, since gastric acidity does not act in the total
elimination of this bacterium. The pathogenicity caused by STEC strains is mediated by
genes expressing shiga toxins (Stx), genes present on the pathogenicity island of the locus of
enterocyte effacement (LEE), and other genes of interest that encode virulence factors.
Among these the major virulence factors attributed to STEC disease pathogenesis is phage-
encoded shiga toxins (Stxs)( Norman et al., 2012).
STEC strains, in addation to its stx also have virulence factor which is Lipopolysaccharide
(LPS), as a major component of the outer membrane. It consists of three distinct regions:
lipid A(main driver of inflammatory responses), core oligosaccharide, and O-specific
polysaccharides (O-antigens). O-antigens are highly immunogenic and one of the most
variable bacterial cell constituents, with variation in the types of sugars present, their
arrangement within the O-unit, and the linkages between O-units that contribute to O-
antigen diversity and are the basis for serotype diversity; there varation is not only exists
between different bacterial species, but also between individual clones within a single

10
species, therefore, they are targeted by the adaptive immune system(Wang et al., 2016). This
high variability is important charaterstic for researchers to tracing infection chain and risk
assessment of STEC strains isolated from patients or food for human consumption based on
their serotype (Geue et al., 2002).

Table 1: Pathogenicity genes of interest in shiga toxin serotypes and their promoter
function in cells

Pathogenicity genes and their promoter function in cells adpted from ( Castro et al., 2017).

11
The infection mechanism of action of the STEC strain begins by binding of the bacteria to
the target cell. Once adhered to intestinal epithelial cells, STEC strain initiate to produces and
releases shiga toxin 1 and/or shiga toxin 2, both, or subtype stx1 (stx1a, stx1c, and stx1d) and
stx2 (stx2a, stx2b, stx2c, stx2d, stx2e, stx2f, and stx2g) into the extracellular milieu and they
consist of a similar structure, an A-subunit associated with five identical B-subunits (Zhu et
al., 2007).
The produced and released shiga toxin passes across the intestinal epithelium and delivers to
its receptor which is globotriaosylceramide (Gb3), by circulating neutrophils. In this cause
the B-subunit which is responsible for the binding of the toxin to the host cell
globotriacylceramide (Gb3) receptor present on the surface of host cells triggering
internalization of the toxin, leading to the internalization of the toxin. However, there are
reports that Stx also can enter Gb3 negative cells through retrograde trafficking and bind to
kidney and brain endothelial cells through globotetraosylceramide (Gb4) surface glycoplipid
receptors. This indicates that Gb3 likely is not the sole receptor involved in Stx-mediated
disease pathogenesis (Ho et al., 2013).

The shiga toxin is formed by a subunit A, is internalized in the cell and is transported to the
Golgi complex and endoplasmic reticulum. This toxin exerts toxic action and an
enzymatically active N-glycosidase that inhibits protein synthesis of cells by cleavage of an
adenine base from the 28S rRNA component of the eukaryotic ribosomal 60S subunit,
causing cell death and also induce programmed cell death (apoptosis) ( See Figure 2)
( Abdullah et al., 2014; Castro et al., 2017).

12
 Fig
ure 2: Mechanism of action of the shiga toxin. (1) Binding of the bacterium to the gb-3
layer of the cell. (2) Production of the shiga toxin. (3) Stx is transported to the Golgi
complex. (4) Endocytosis of the toxin in the cell. (5) Clathrin-enveloped vesicle formation.
(6) The toxin is transported to the Golgi complex. (7) Vesicle breaking and separation of
pentamer B from the toxic A1 fraction. (8) Action of the A1 portion on the rRNA in the 28S
portion, acting as N-glyocsidase, replacing an Adenine moiety. (9) Inability to translate the
RNA tape. (10) Cell death

3..2. Host immune response against STEC Strain Infection

The general procces of STEC to infect host cell can be described as follows: i) colonization
of the gut, ii) virulence factors effect on the host cell and iii) disease caused by the virulence

13
factors. Therefore the host cell designed their defence mechanism. Different studies results
provide evidence support the hypothesis that infections with STEC( Stx) can suppress the
development of specific cellular immune responses by targeting peripheral blood and
intraepithelial lymphocytes (IEL) Once the STEC strain attached to host cell surface, the
innate (natural/ non clonal/ non adaptive) immune system of host cell defense against STEC
infection is activated as first phase of defence using their pathogen pattern recognition
receptors(Toll-like receptors (TLRs)(Hoffman et al., 2006).
At this phase of defence the pathogens that manage to penetrate the body’s physical barriers
(skin, mucous membranes and enzymes) are limited to spread through the host’s physiology:
skin pH or proteases in the saliva. When the pathogens recognized by phagocytic cells
through pathogen-associated molecular patterns, leading to intracellular signaling and
ultimately resulting in the production of cytokines and chemokines and activation of the
adaptive immune system. One of the best-known PAMPs is endotoxin (LPS), which is
responsible for activating innate host defense mechanisms in gram-negative infections
(Judith et al., 1997).
The innate immune system consists of cells and complement that are activated by microbial
structures. Cells in the innate immune system, such as monocytes, macrophages, dendritic
cells and neutrophils, express a variety of receptors (pattern recognition receptors) that
enabling to recognise microbial structures (Iwasaki and Medzhitov, 2010).
Toll-like receptors (TLRs) are a class of proteins that play a key role in the innate immune
system by recognize microbial/pathogen-associated molecular patterns and they are usually
expressed on  macrophages and dendritic cells, that recognize structurally conserved
molecules derived from microbes. Once these microbes have reached physical barriers such
as the skin or intestinal tract mucosa, they are recognized by TLRs, which activate immune
cell responses (Roger et al., 2008). Different studies report indicates that 10 TLRs have been
identified in humans and 13 TLRs in mice, among these TLR 4 and TLR 5 recognize specific
microbial components such as Gram-negative bacterial lipopolysaccharide and flagellin
respectively. They are located at the cell surface of phagocytic cells, but may also be
recruited into phagosomes (Nasim et al., 2017).
The activation of TLRs leads to activation of the immune system, resulting in the release of
immune mediators and immune cell activation that clear the bacterial STEC infection.

14
Different signal transductions are involved for immne system activation aganst STEC
infection. One of the most important signalling cascades that involve activation process to
clear a microbial pathogen is the interferon-gamma (IFNγ) signal transduction pathway
(Proulx et al., 2001). Proinflammatory cytokines, including IFNγ, are secreted into the
extracellular environment by macrophages, natural killer T cells, and activated T cells
following STEC infection resulting in the activation of up to 2000 IFNγ-stimulated genes in
recipient host cells that together mount the host defense against pathogenic microbes (Ho et
al., 2013)
When the innate immune system cells are unable to eradicate the STEC pathogen, the
immune system cells (lymphocytes) generate humoral and cell-mediated adaptive responses
to pathogens. The integration between innate and adaptive immunity occurs through
chemical messengers and through direct contact between cells of the innate and adaptive
immune responses (primery and secondary) that cooperation leads to an optimum defense
against the target pathogens (figure 3).

15
 Figure 3: Innate and adaptive immune response aganst Gram negative bacteria (STEC
Strain)

3..3. Prevalence(Occurance ) of Shiga Toxin-Producing Escherichia coli (STEC)

Shiga toxin-producing Escherichia coli (STEC), including O157 and many non-O157
serotypes, are recognized as an important group of food-borne bacteria associated with
outbreaks worldwide. They cause human serious illnesses ranging from common diarrhea to
hemolytic-uremic syndrome (HUS), a life-threatening complication and death. Near to 5%–
10% of people with STEC infection have a chance to develop hemolytic-uremic syndrome
(HUS), from those who develop HUS will die or have permanent renal failure, and up to
50% of those who develop HUS will develop some degree of renal impairment(Croxen et al.,
2013).
STEC is a global problem, and more than 60 serotypes have been associated with human
disease. Globally STEC causes annually 2, 801, 000 acute illnesses, with an incidence rate of
43.1 cases per 100 000 persons. From those 3, 890 cases of HUS developed and 230 deaths.
Among those, a total of 10, 200 cases of STEC infections occur in Africa with an incidence

16
rate of 1.4 cases per 100,000 people annualy. STEC O157:H7 contributes 10% to this burden
(Athumani, 2018).
Escherichia coli O26, O45, O103, O111, O121, O145, and O157 are the predominant shiga
toxin–producing E. coli (STEC) O- serogroups implicated in outbreaks of human foodborne
illness worldwide. From these O157:H7 STEC is the most prevalent and a serious public
health problem (hemorrhagic colitis (HC) and HUS in children in many developed and
developing countries (Sang et al 2012).
Because of initial predominance in human clinical infection to date, STEC prevalence studies
and detection (culture and molecular) method development and optimaized have been
focused primarily on E. coli O157:H with little attention to and resultant under estimation of
the risks posed by non-O157 serogroups(Monaghan et al.,2011).
However, some prevalence studies show an increasing number of outbreaks of non-O157
STEC(O26, O121, O103, O111 and O145 - most predominant non-O157 STEC globally)
(related infections have been confirmed. Outbreaks involving non-O157 strains worldwide
are more common in Europe, Australia, and Latin America. European Union data indicate
that the most frequent serotypes in infections reported during 2007 to 2011 were due to O157
(49% to 76%), O26 (7% to 11%), O103 (3% to 4%), O91 (2% to 3%), O145 (2% to 3%),
O111 (1% to 2%), and O128 (1%). In the United States, from 2000 to 2010 the incidence of
non-O157 serotype infections increased from 0.12 to 0.95 per 100,000 inhabitants, while
O157 infections fell from 2.17 to 0.95 per 100,000 inhabitants during the same period(Castro
et al., 2017).
Overall STEC strain isolates have been reported in more than 30 countries on 6 continents.
Reports on isolation of pathogenic STEC O157:H7 isolation has been reported from all
regions of the African continent (east, west, south, north and central) from humans, animals,
food products and the environment. Out of 30 reviewed cases, 10 (33.3%) come from human
patients and the remaining 20 isolations (66.7%) belong to food stuffs and animals
(Majowicz et al., 2014). In Africa all virulence genes, such as stx1, stx2, eae and ehxA genes,
have been detected in humans, animals, food products and the environment. The most
dominant combination was stx1 + stx2 and Cattle are the most common source of STEC
O157:H7, as shown in Table 2 (Athumani et al., 2018).

17
Table 2: STEC O157:H7 virulence factor combinations from studies in Africa

In Ethiopia also the risk of disease due to pathogenic E.coli is high because of raw meat
consumption traditionally. However there is an argument about the occurance of pathogenic
Escherichia coli in foods of animal origin (meat, milk) due to many reasons like illegal
slaughtering of animals in open fields, unhygienic slaughter practices in the abattoirs. The
overall prevalence of the organism in meat and milk is 16% and 26% respectively. This
high level of prevalence specially in milk is may be due to contamination of milk with cattle
feces during milking because cattle are the reservoirs of the organism(Abdissa et al., 2017).
In Ethiopia, there were only very few studies conducted by some researchers to determine
the occurrence and prevalence of STEC such as E. coli O157:H7 in animals, animal products
or people in different areas of the country to generate information and create awareness in the
public and formulate preventive measures along food production, processing, and
distribution. The studied report indicated that the prevalence of E. coli O157: H7 in feces,
intestinal mucosal swab and skin swab samples collected from cattle slaughtered at
processing plants and at the retail shops 14.2 % E. coli O157:H7 was isolated from 446
samples. Research conducted in Addis Ababa, Debre Zeit municipal abattoir and Modjo
export report showed that the prevalence of E. coli O157:H7 is 10.2%. and also research
conducted in the eastern part of Ethiopia (Dire Dawa and Haramaya), prevalence of E. coli

18
 O157:H7 as a contaminant of meats of ruminants was recorded from 2.55% up to 30.97%
and 62.5% E. coli from beef samples collected from Mekele Municipal abattoir and meat
retailers (Ayalew and Amare, 2018).

 Diagnosis/ Detection Mechanism of STEC Strain

There are a number of difficulties associated with the detection of STEC infection due to it
number flexibility, large numbers of STEC at the early stages of infection and dramatically
dropping of its number as the disease progresses. In addition, because of the diversity of
shiga toxins in nature and their broad range of hosts, detection and differentiation of these
toxins or outer membrane of O157:H7 and other new and emerging serotypes of non-O157
“big six” (O26, O45, O103, O111, O121, and O145) (Hannah et al., 2009) by using very
rapid, prompt, accurate, specific, sensitive and require minimal specimen volumes is
important for effective and timely outbreak responses and prevent unnecessary invasive and
expensive surgical and investigative procedures or administration of antibiotic therapy which
may be contraindicated for STEC infection (James and Paton, 1998).
Historically substantial progress has been made in the development of detection assays for
STEC strains detection and identification based on the presence of Stxs and their
immunogenic outer membrane (LPS) parts with differs in complexity, speed, sensitivity,
specificity and cost in human, animals and environmental samples(Castro et al., 2017,
Skinner et al., 2013). Some of them are stool culturing and isolation which is based on
inability to ferment sorbitol within 24 hours on sorbitol-containing agar isolation media,
polymerase chain reaction (PCR) using a multiplex system, which is based on many types
and subtypes of Stx at the same time, quantitative real-time PCR (qPCR), which measure the
presence of a given gene in a sample. Reverse Transcription qPCR (RT-qPCR) ,which
measures gene expression, other method has been developed using monoclonal antibodies
that can bind to the toxins and differentiate them based on their immunological properties.
However each diagnostic or detection methods have their own advantages and limitations so,
it is important to use them in a complimentary fashion for robust detection and differentiation
(Vallieres et al., 2013).

19
 The main limitation of these developed detection methods are time requirement (24–48 h for
culture), require high specimen volumes, difficulty in isolating DNA from sample (for PCR)
and low sensitivity (for immunodiagnostic) (William et al., 2016). Generally an ideal
detection or diagnostic method fulfill five premier requirements such as high specificity
(detecting only the bacterium of interest), high sensitivity (capable of detecting as low as a
single live bacterial cell), short time-to-results (minutes to hours), great operational
simplicity (no need for lengthy sampling procedures and use of specialized equipment) and
cost effectiveness. For example, culture takes long time to give the results. On the other
hand, PCR, antibody-based techniques and biosensors offer shorter waiting time, but these
require the use of expensive reagents and sophisticated equipment which make the method
expensive(Priyanka,2017).

3..1. Culture Based Methods for Detection of Shiga Toxin-Producing E. coli

Culture based methods has been the oldest methods for detecting the microorganisms, even
the pathogenic strains. For many years, Sorbitol-MacConkey Agar Culture (SMAC)
detection method has been the most commonly used for isolation of STEC strain especially
in Northern America and Europe and O157 and O157:H2 were detected
predominantly(March et al., 1986). STEC O157:H2 E.coli can be easily distinguished from
other most commensal E. coli strain by their inability to ferment sorbitol within 24 hours of
incubation at 35°C on a selective and differential media, such as sorbitol-MacConkey agar
(SMAC), cefixime tellurite-sorbitol MacConkey agar (CT-SMAC), or CHROMagar O157. In
this cause E. coli ferments sorbitol within 24 hours and produces β-glucuronidase (Linda et
al., 2010). These metabolic characteristics are important for the differentiation of the STEC
group, which, unlike most other E. coli strains, grows little or not at all at 45.5 °C, while
O157 species do not ferment sorbitol (during the first 24 h) which are colourless on SMAC or
CT-SMAC (Sata et al., 2003) and are pink on CHROMagar O157 and do not produce β-
glucuronidase (Gouali, 2013).
Both CT-SMAC and CHROMagar O157 are more selective than SMAC, which increases the
sensitivity of culture for detection of O157 STEC. The sensitivity of SMAC is limited to
recognize non-fermenting colonies against the background of other organisms on the plate

20
and its other false positive results due to the emerging serotypes of sorbitol fermenting non-
O157 and O157 STEC.The drawbacks of the SMAC agar can be overcome by the use of
chromogenic medium for STEC isolation which has increased specificity and sensitivity
(Growther and Andrew, 2016). Representative STEC culture-based tests results are shown in
(Figure 4).

Figure 4: STEC isolation from various selective media. (A) Cells from enrichment broth
are plated on CT-SMAC. (B) Suspect colonies appear as pale on CT-SMAC and steel blue on
NT-Rainbow Media, and non-O157 STECs appear as pink colonies on NT-Rainbow. (C)
Suspect STECS expressing b-galactosidase and hemolysin are indicated by blue colonies
with a zone of clearing on Sheeps blood agar. (D) Typical non-O157 STECs are shown
growing on CHROMagar and appear as blue colonies (William et al., 2016).

Table 3: Different typepes of medium and methodologies (official and unofficial) used to
detect STEC.

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 Enrichment: mBPWp - modified buffered peptone water with pyruvate; mTSB - modified tryptone soya broth; IMS -
immunomagnetic separation; mEC - EC broth (modified) with novobiocin; BPW - buffered peptone water; TSB -
tryptone soya broth. Cultura Media: L-EMB - eosin methylene blue (levine); SHIBAM - STEC heart infusion washed
blood agar with mitomycin; mRBA - modified rainbowR agar; SBA - sheep blood agar; TBX - tryptone bile agar with X-
glucuronide; CT-SMAC - MacConkey agar with sorbitol, cefixime, and tellurite; SMAC - MacConkey agar with sorbitol;
NT-RA - rainbow agar O157; mSBA - sheep blood agar modified; CCV-TBX - tryptone bile agar with X-glucuronide,
cefixime, cefsulodin, and vancomycin; wSBAm - washed sheep blood agar containing mitomycin; SMAC-BCIG -

MacConkey agar with sorbitol and chromogen 5-bromo-4-chloro-3-indolyl-b-D-glucuronide. (Castro et al., 2017).

However, some studies were conducted in 2013 and their result indicates that O157:H7
strains capable of fermenting sorbitol were identified, as well as an outbreak of a sorbitol-
positive O157:H7 strain. This demonstrates the limitation of detection based only on
bacterial metabolism (Feng et al., 2017). Culture-based methods (e.g. sorbitol-
MacConkeyagar) remain the gold standard test, cost-effective and given the importance of
identifying viable bacterial isolates for typing. For this reason, there has been increased
development and use of agars which also select for non-O157 STEC (Verhaegen et al.,
2016). However, the biggest drawback in the culture-based method are laborious, time
consuming, and exhibit clear limits in sensitivity for STEC detection, false negative results
due to emerging serotypes of non-157 STEC and sorbitol fermenting O157:H- STEC limit
the utility, it is recommended that laboratories supplement culture-based approaches with
other assay types(Parsons et al.,2016).

3..2. Tissue Culture Cytotoxicity Assays (VCA) for Detection of STEC

The Vero cell (derived from African green monkey kidney) and HeLa cell lines(lacks Gb4-
less sensitive to Stx2e) have been developed to detect Shiga toxins because they have high
concentrations of globotriaosylceramides Gb3 and Gb4 (target receptor for Stx2e)receptors
for STEC (stxs) due to that they are very sensitive to shiga toxin and use the toxins to entre in
to eukaryotic cells (Li et al., 2015).
The Vero cells assay is performed by transfer of STEC cell-free supernatants to tissue culture
monolayer’s of Vero cells for incubation and observed their typical cytopathic effect. In this
cause the presence of Stxs, Vero cells take a round shape and disconnect from one another
and the degree of cytotoxicity can be estimated within 24 and 48 h by using microscope but

22
 the cytotoxicity effect may be due to other toxin or bacterial product, therefore it is
important to perform a neutralization assay( which avoid VCA specificity problem) with
specific antiserum( anti-Stx 1 and anti-Stx 2 antibodies) to confirm that the cytopathic effect
is because of the production of Shiga toxins(William et al., 2016).
Many studies have shown that the Vero cells assay is very sensitive, However, several
constraints have limited its routine used in most clinical diagnostic laboratories because it
requires a highly skilled technician with tissue culture technique to maintain the cell lines as
well as monoclonal antibodies for confirmation of shiga toxin, the availability of cell
monolayer’s and specific antibodies, requires cell culture facilities and time consuming to
obtain the results (48-72) hours (Gerritzen et al., 2011). To avoid the limitation concern with
tissue culture cytotoxicity assays, non-culture assays that can detect the shiga toxins
produced by STEC have been first introduced in the United States in 1995. This assay can
detect all serotypes of STEC with quickly to obtain the results than culture (Hannah et al.,
2009).

3..3. Immunological Methods for STEC strain Detection

The first immunoassay was developed by Rosalyn Sussman Yalow and Solomon Berson in

the 1950s. Immunological detection of STEC strains are mainly based on the binding of
antibody-antigen interactions, whereby a particular antibody (monoclonal or polyclonal ) will
bind to its specific antigen immobilized on membranes and horse radish peroxidase for
detection or using monoclonal or polyclonal antiserum against shiga toxin produced by
STEC strain after an enrichment step(Peruski, etal., 2003). pAb is more important to develop
a better assay than monoclonal antibody because polyclonal antibody usually have higher
reactivity than mAbs due to the polyvalency (multiple epitopes to react with) versus the
monovalency of a mAb(He et al., 2013).
Immunological detection has been a widely used method to detect STEC antigen in bacterial
cultures, food samples and fresh or frozen stool samples. Currently, several different types of
commercialized immunological diagnostic assay kit with various formats have been
developed for the detection of Stx1 and Stx2 producing E.coli and other pathogenic
antigen/stx. Such diagnostic kits are immune-preciptate, immunodiffustion, electroimmuno

23
diffusion, Radioimmunoassay (RIA), Counting Immunoassay (CIA), Enzyme-linked
immunosorbent assays (ELISA), Immuno agglutination, latex agglutination
Fluoroimmnoassay (FIA), Chemiluminescenceimmunoassay(CLIA) and others (Darwish,
2006).
Among these Enzyme Linked Immunosorbent Assay (ELISA) have been reported as popular
and the most commonly used immunological methods for the detection of STEC strain with
high sensitivity and specificity. The detection mechanism is performed by capture antibody
which is linked to an enzyme, or by a secondary enzyme- linked antibody targeting the
capture antibody. Then, by adding the enzymatic substrate, a visible signal proportional to
the amount of antigen is produced There are different types of enzymes can be used in
ELISA, some of the most commonly used enzymes that conjugate with secondary antibodies
are include horseradish peroxidase (HRP), alkaline phosphatase and beta-galactosidase (law
et al., 2014).
During the past decade, a number of enzyme-linked immunosorbent assays (ELISAs) have
been developed with diferent format for the detection of Stx1 and Stx2 in fecal cultures.
There are four ELISA methods described in (figure 5) such us; Direct ELISA
(dELISA),Indirect ELISA (iELISA) , Competitive ELISA (cELISA) and Competitive ELISA
(cELISA).

Figure 5: Enzyme Linked Immunosorbent Assay (ELISA) methods with different format

24
The disadvantage of immunodiagnostic assay kit is the possibility of cross-reactivity leads to
false positive and attributed to a lack of specificity of the assay (ELISA)9 (parsons et al.,
2016). Moreover, in addition to (ELISA), newer techniques such as flow cytometry
(comparison of genetic fragments) assay have been developed for the detection of the 6
majors non-O157 STECs. This assay is fast, specific, and efficient (Hegde et al.,2012). Other
methods are also being used, such as slide and latex agglutination kits for the detection of
STEC serotype like O157, which are faster and easier than other methods. Slide agglutination
test is the cheapest, simple, rapid and easy to perform and it needs few minutes only to
perform the test, due to these characteristics slide agglutination based STEC detection is
routine used in most clinical diagnostic laboratories (figure 6) (Hatch and Scalarone, 2013).

In addation, test ube agglutination test is also used to quantitate the amount of serum
antibodies against a STEC (Stxs or LPS) by serially diluting the serum and then mixing the
diluted sera with constant quantity of STEC antigens. In this cause the titer reflects the
concentration of antibodies in the serum; the higher titer indicates the more concentration of
antibodies (Hajra et al., 2007).

Figure 6: Slide Agglutination Test Result

25
3..4. Molecular Based Detection of Shiga Toxin-Producing E. coli

Besides phonotypical and immunological assays of STEC strain detection DNA-based

methods represent a rapid and robust alternative in the principle of detecting virulence factor
genes of STEC. In recent years, many diagnostic laboratory have switched to polymerase
chain reaction (PCR) as a diagnostic tool for detection and distinguish between stx1, stx2,
and other putative virulence gene (eae, ehxA) in pathogenic STEC strain( Haugum et al.,
2014).
Polymerase chain reaction (PCR) is the most common methods for detection of virulent
toxins and other virulence markers coding gene. Currently, there are a range of molecular
techniques such as conventional PCR, multiplex PCR, quantitative PCR (qPCR) as well as
Real time PCR (RT-PCR) that applicable for STEC detection. Of these, multiplex PCR and
real-time PCR are the most popular. Amplification of target genes of STEC strain DNA
extracts from feces is less successful than from pure cultures, therefore to improve its
sensitivity, careful preparation of the sample (DNA) is important(Quiros et al., 2015)).
Crude lysates or DNA extracts obtained from single colonies, as well as mixed broth
cultures, colony sweeps, or direct extracts of feces or foods can be used as DNA templates
for PCR and its amplification products are usually detected by ethidium bromide staining
after separation by agarose gel electrophoresis. The occurrence of real time PCR makes it
possible for simultaneous quantification and detection of E. coli (Asmelash, 2015).

However, detection of stx is not always representing STEC because stx can be located in the
genome of temperate phages found in the samples as free particles; this could explain the
numerous reports of positive stx detection without successful STEC isolation. A range of
molecular techniques such as conventional polymerase chain reaction (PCR), RT-PCR, PCR
coupled to mass spectrometry, and isothermal nucleic acid amplification, have been
employed in the detection of gene targets associated with STEC strains(Silva et al., 2017).
Stx specific PCR assays are specific, sensitive and less time-consuming. However, PCR
detect the toxin gene sequence, not the toxin itself and the presence of a gene does not
indicate whether (or by how much) a gene is actually expressed. As stx genes are
bacteriophage-encoded, they are acquired by horizontal gene transfer from a bacteriophage,

26
and Stx expression is largely dependent on phage replication within the bacteria (Fagerquist
and Zaragoza, 2015).
For STEC identification, several genes associated with virulence are commonly used
including rfbE encoding the O157 polysaccharide (antigen O), flicC encoding H7, specific
flagellar antigen, eaeA encoding intimin, hlyA encoding hemolysin and uidA (gusA)
encoding β-glucuronidase. Genes found in the O-antigen gene clusters that show genetic
variability among the different serogroups include the wzx (O antigen flippase) and wzy (O
antigen polymerase) genes, and PCR assays targeting these genes have been developed to
identify different E. coli serogroups (Liu et al., 2015).

Table 4: Detection methods by PCR of STEC-associated target genes

27
 In general, the major advantages of molecular (PCR) based are that the process is rapid,
sensitive and specific than the culture based methods and immunoassays. However, there are
certain disadvantages that make it necessary to develop better methods. The difficulties
include cell lysis and nucleic acid extraction, cross-contamination and failed reactions due to
the presence of inhibitory substance or competing DNA from the non-target cells. Other
disadvantage are not able to differentiate between the live and dead cells, chances of
generating false positive signal due to binding to non-specific double-stranded DNA
sequences(Parsons et al., 2016).

3..5. Other emerging technologies for STEC strain detection

Conventional pathogen detection methods, such as microbiological and biochemical

identification, are time-consuming and laborious while immunological or nucleic acid-based
techniques require extensive sample preparation and sophostification equipment and reagents
(Ahmed et al., 2014). Due to this, other improved detection methodology /technology have
been developed, such as biosensors, DNA microarray, nano-biotechnology and others.
Biosensors are the devices for pathogen detection that generally consist of three elements,
which are a biological capture molecule (probes and antibodies), a method for converting
capture molecule – target interactions into a signal and an output data (Chen and Cheng,
2017).
The major advantage of the biosensors is that these can detect the pathogens at low detection
limits with high specificity and sensitivity, but the biosensors will require highly specific and
expensive instruments, with compatible computer software, to give accurate results. Hence,
these methods may not be always cost-effective (Ahmed et al 2014).

3.3. Development of immunodiagnostic kit for STEC Detection Using Antibodies

3.3.1. Antigenic (immunogenic) parts of STEC selection, isolation and purification for
antibody production

An antigen is any molecule that is identified as non-self by components of the immune

system that able to induce an immune response, including production of antibody via the

28
humoral or cellular immune response. There are three characteristics that a substance/antigen
must have to be immunogenic: foreignness, high molecular weight (more than 6000 dalton)
and chemical complexity. The cellular immune response against antigen is mediated by T
lymphocytes and can not be transferred from one individual to another by transfusion of
serum. Whereas humoral immunity (targets extracellular antigens) involves soluble proteins
found in serum (antibodies) that can be transferred to a recipient when serum is transfused
(Osato et al., 1972).
B-lymphocytes use membrane IgM (mIgM) to bind antigen in native form. In this cause
mIgM and antigen molecules makes a complex which is then taken into the cell by receptor
mediated endocytosis. This endosome then digest the antigen into small peptides by fuses
with a lysosome. The endolysosome fuses with a vesicle containing class II Major
Histocompatibility Complex (MHC II) molecules and the then peptide antigens are bound by
MHC II. This MHC II/antigen complex then expressed on the plasma membrane of the B-
lymphocyte. The T cell receptor of a T helper lymphocyte then binds with the MHC
II/antigen and the T cell secretes cytokines that signal the B-lymphocyte to divide, proliferate
differentiate and secrete antibodies (Wieczorek  et al., 2017).
Pathogenic E. coli STEC strains can be classified into serotypes by antigens in their cell wall
O antigen: part of lipopolysaccharide layer, in their flagella: H antigen and their Capsule: K
antigen, that can elicit an immune response in animals. Due to the presence of different
sugars and sugar linkages O antigen under LPS is an immunodominant molecule that is
important for the virulence and pathogenesis of many bacterial species (Zhang et al., 2013)
and it is the most specific and variable within serotype of STEC strain. The O antigen, which
consists of many repeats of an oligosaccharide unit, is the outer component of LPS in the
surface of Gram-negative bacteria. Due to the presence of different sugars and sugar
linkages, it is one of the most variable cell constituents which plays an important role in
bacterial evasion of host defense systems(Beutin et al.,2007).
Lipopolysaccharide (LPS) is an important structural component of the surface of gram
negative bacteria which constitutes about 75% of the surface and 5–10% of the total dry
weight of gram negative bacteria and it is recognized by immune cells as a pathogen-
associated molecule that activates immune system vie triggering cytokine( TLR-4) release
from cells of different origin(Sohrabi et al.,2011). Lipopolysaccharide (LPS) is a complex

29
structure and which consists of of three parts such as lipid A region, core oligosaccharide and
repetitive polysaccharide designated as “O” antigen. Lipid A is highly conserved
hydrophobic portion of the Gram-negative bacterial outer membrane and contributes to the
toxicity of the LPS (Rezania et al., 2011). While the “O” antigen carbohydrate chain is a
polymer of immunogenic repeating oligosaccharides (1–40 units), which differs between
species and is a major contributor to the serological specificity of bacteria and the other
component is core region which is phosphorylated nonrepeating oligosaccharides (figure 7)
(Rezania et al., 2011).

Figuer 7: Structure of a lipopolysaccharide of STEC strain

The genes that encode for O-antigens are located on the chromosome in a cluster designated
as the O-antigen gene cluster (O-AGC) between housekeeping genes (UTP-glucose-1-
phosphate uridylyltransferase) and gnd(6-phosphogluconate dehydrogenase) at the 3′ end in
E. coli. These gene are classified into three main classes: (i) genes for biosynthesis of
nucleotide sugars; (ii) genes for transfer of sugars from their respective nucleotide sugar
donors to build O unit; (iii) genes for processing steps in the conversion of the O unit and
carrying out specific assembly to form the O-antigen as part of LPS, including flippase gene

30
 (wzx) and polymerase gene (wzy) (Beutin et.al, 2007). Escherichiacoli serogroups O5, O15,
O26, O45, O55, O76, O91, O103, O104, O111, O113, O118, O121, O123, O128, O145,
O146, O157, O165, O172, and O177 are the O-antigen forms of the most clinically relevant
Shigatoxin producing E.coli (STEC) serotypes (Sanchez et al.,2015).

Therefore to design specific immunodiagnostic kit for STEC detection using antibody against
STEC, isolation, purification and characterization of LPS is important step. However,
contamination with capsular polysaccharide, nucleic acids and outer membrane proteins like
peptidoglycan which potentially interfere with downstream process is the main problem with
LPS purification procedures which hinder its reliable application in most downstream
immunological and biological experiments (Carol et al., 1999). Numerous methods have
been developed for isolation and purification of LPS of STEC among which the Hot-Phenol
method is a most frequently used with its limitation (cancerous, tedious and poisonous
nature). However methanol-chloroform method is easy, less expensive (economical), quick
and safer with its drawback (contamination of LPS yielded with capsular polysaccharide,
nucleic acids and outer membrane proteins, which are introduced to the final purified LPS
during extraction and purification than Hot-Phenol method of LPS extraction (Kalambhe et
al., 2017). However, these contaminant protines are removed by methanol-chloroform
treatment because they aresoluble in methanol-chloroform but LPS is not soluble.

3.3.2. Antibody (monoclonal and polyclonal) Development

To develop immunodiagnostic assay kit, antibodies production is important scheme either

against a single antigen or antigens associated with a specific analyte, pathogen, or disease
condition. For successful generation of antibodies depends on B-lymphocytes to bind,
process and present antigen to T helper lymphocytes, that leads generate signals the specific
B cells that have been stimulated by the antigen binding to the B cell’s antigen-receptor to
differentiate into antibody-producing cells (plasma cells) in the lymphoid organs (spleen,
lymph nodes, among others)(Leeaars et al., 2005) (see figure 8).

31
Figure 8: Antibody development (B-cell proliferate in to plasma and memory B-cell)

After primery immunization naive B cell are stimulated to proliferate and differentiate in to
either effector cells (plasma cells) to combat the current antigen, or memory cells to address
future antigen exposure. After the selected animals injected with the immunogene, specific
antibody begine to generated in the serum withine 5 to 7 days of immunized and their
concentration is continuous to rise and peaks with 10 to 14 days and after which it decreases
Following the initial immunization, booster injections can be administered, which is
important to continue exposure of antigens to B cells. Once the antigen in a host is
neutralized, a small number of activated B cells will differentiate into memory cells, and
most will die by apoptosis (Pappas et al, 1994).

32
However, the lag or adaptaion perod for specific antibody production is short and the peaks
of antibody production (secondary antibody response stimulation) occurs 7 to 14 dayes after
boosting less amount of antigen immunized and their concentration sustained for along
period of (Leenaars et al., 2005).
Antibodies (or immunoglobulin) are a family of proteins of the adaptive immune system that
defend the host by binding to antigens.and they have a similar structure (constant region) in
common that determines the functionality of the antibody and the variable region are
responsible for binding the respective antigens Each antibody containing four polypeptide
chains: two identical heavy chains (H) and two identical light chains (L) thus providing at
least two sites capable of binding an antigen. Heavy chains are connected to each other by
two and above disulfide bonds, whereas each light chain is connected to a heavy chain by
one disulfide bond (Janeway et al., 2001). The amino (N) terminus of a light and heavy
chain contains the hypervariable amino acid region, or the "Fab" portion of the antibody
molecule, whereas the carboxylic acid (COOH) terminus of both heavy chains composes the
crystallizable (Fc portion of the antibody). The V regions of H and L chains comprise the
antigen-binding sites of the immunoglobulin molecules (See Fig.9)( Kirkham et al., 1994).

Figure 9: Structure of an Antibody (immunoglobine) Molecule: The antibody is composed

of two light chains and two heavy chains. The variable regions are located at the amino acid terminal
end of the molecule. The light chain is composed of one variable region and one constant region. The

33
heavy chains are composed of one variable region and three constant regions. The hinge region
allows flexibility in the molecule for antigen binding. The antigen binding sites are specific and are
represented by the complementarity-determining regions (CDR) regions. The heavy and light chains
are connected via disulphide bonds, and there are disulphide bridges at the hinge region also between
the two heavy chains. Disulphide bonds are also present in the constant and variable regions.

Antibodies are produced and purified in two basic forms for use as reagents in immunoassays
such as polyclonal and monoclonal. Antibodies those produced and secreted by a single clone
of B lymphocytes are called monoclonal antibodies, and while those that have heterogeneous
immunological response to an antigen and produced by a mixture of different cell lines of B
lymphocytes clones (originate from common stem cells), termed as polyclonal antibodies
which is serum from an immunized animal will contain numerous antigen specific antibody
clones. Both products have become important tools in fundamental immunological research,
immunohistochemistry, diagnostic testing, and vaccine quality control (Lenars and
Hendriksen, 2005).
In the production of polyclonal and monoclonal antibodies, a number of critical steps are
developed, such as preparation of antigen samples, selection of the animal species with their
injection sit, selection and preparation of the adjuvant, post-injection observation, and
collection of the antibodies (Boer and Heuvelink, 2000) . In the cause of polyclonal antibody
production, an antigen/adjuvant conjugate is injected into an animal of choice to initiate an
amplified immune response. After a series of injections over a specific length of time, the
animal is expected to have created antibodies against the conjugate. The polyclonal
antibodies are acquired from a host via blood sampling, which involves collection from the
host every 2 weeks and then purified by centrifuged the collected blood to obtain the
antibody of interest (He et al., 2013)
Therefore, a polyclonal antiserum can be obtained within a short time (4-8 wk) with little
financial investment. Whereas it takes about 3 to 6 month to produce MAbs. However,
monoclonal antibodies are derived from a single cell line through by fusing antibody-
secreting spleen cells from immunized mice with immortal myeloma cell to create
monoclonal hybridoma cell lines that express the specific antibody in cell culture supernatant
(Peruski et al., 2003)(see figure 9).

34
Figurer 10: polyclonal and monoclonal antibody production scheme

Figu
re 11: Immunoglobulin diversification and B cell development: B cell development as a

35
function of immunoglobulin rearrangement and modification. After birth, B cell development
begins in the bone marrow and which is independent of antigen stimulation. With
development, the fate of the B cell becomes increasingly dependent on its response to
antigen. Immature B cells leave the bone marrow and begin to express IgD. They recirculate
through the blood, the secondary lylmphoid organs and the bone marrow. Encounter with
cognate antigen can cause the cell to become a memory B cell or a plasma cell. However, the
patients with X-linked agammaglobulinemia (XLA). and have difficulty making immature B
cells and IgM. Patients with hyper IgM syndrome (Hyper IgM) are unable to class switch.
Patients with selective IgA deficiency (IgAD) or common variable immune deficiency
(CVID) can class switch, but have difficulty becoming plasma cells or memory B cells.

The first immunoglobulin class to be generated by antibody-producing B cells is IgM, which

is the most efficient immunoglobine (900,000 daltons MW) in agglutination reaction and a
pentavalent with high affinity for antigen epitopes as primery response. The IgM response
decreases with time and is replaced by a second, high avidity immunoglobulin class termed
IgG(150, 000 dalton MW) antibodies as secondary immune response and others are
generated with different concentration. Their concentration of IgG, IgA, IgM, IgE nd ,IgD
in serum is approximately ,10-16mg/mL, 1-4mg/mL, 0.5-2mg/mL, 0.00001-0.0004mg/mL
and 0-0.4mg/mL and their total concentration in serum in presentage is 80%, 10-15%, 5-
10% , <0.002% and <0.2% respectively (Schroeder et al., 2010).

36
 4. MATERIALS AND METHODS

4.1. Bacterial isolates and bacterial culture condition

In this study, 40 E. coli strains among them 10 STEC strains and 10 entropathogenic, 5
EHEC remaining pathogenic E. coli strains and 10 non-pathogenic E. coli strains isolates and
5 non E. coli strains species ( Salmonella, Shigella, and Vibrio and others), available as
glycerol stocks in the laboratory of the supervisor and also to be requested from local and
international institutions having culture collection such as EPHI, will be collected. EMB,TSA
and Mac Conkey Agar will be used to culture E. coli strains and incubated aerobically
overnight at 37 oC. Other non E. coli bacterial strains will prepare by transferring 5 ml Luria–
Bertani (LB) broth and incubated overnight at 37oC and used in this study for LPS
preparation for polyclonal antibody production.

4.2. Preparetion of antigen (Lipopolysaccharide-O antigen) of STEC strain by

chloroform-methanol method

In this study Lipopolysachharide (LPS) will be extracted by Chloroform-Methanol method as

per (Kalambhe et al., 2017) with some modification. About 250 ml overnight grown broth
cultured of STEC strain will be transferd in to centrifuge tube (12ml) and centrifuged at 3000
rpm at 4 oC for 30 minutes. The supernatant part will be discarded and the pellet part
transferd in to 2ml centrifuge tube and re-suspended in 1.5 ml of 95 % alcohol by vortexing
and then centrifuged for 10 min at 2000 rpm wiil be conducted and this step will be repeated
three times.
Finally after discared the supernatant part, the pellet part will be dried by placing the tube
inside hood to completely evaporate alcohol. The dried pellet again will be re-suspended in
one ml of 10 % EDTA and it will be sonicated for 20 minits. One ml of saturated
methanol/chloroform (1:2 ratios) will be added to bacterium- EDTA solution. The tube lid
will covered by paraffin and kept on shaker for two hours following 10 minits centrifugation
at 2000 rpm. Three layers will be formed; methanol, left biomass including cell lysate and
chloroform layer from top to bottom.

37
 The biomass layer with cell lysate will be discarded and chloroform and methanol layers
will be separated and poured into 50ml glass beaker to permit complete and quick
evaporation of methanol and chloroform layers. The LPS will be generated as dried pellet.
Finally LPS pellet will be reconstituted in sterilized 1% of PBS (quantity enough to dissolve
a pellet completely) and stored at -20 oC until further used (Kalambhe et al., 2017).

4.3. Determine the purity of extracted LPS by Sodium Dodecylsulphate Polyacrylamide

Gel Electrophoresis (SDS-PAGE) followed by commassie blue staining

In order to determine the purity of the extracted LPS, it will be separated by SDS-PAGE and
stained with commassie blue staining as followed: all required SDS-PAGE equipment’s
(clean dry glass plates, combs, and spacers) will be arranged and gel cassette will be
assembled by following manufacturer instructions. Then 15% of resolving gel or separating
gel solution will be transferred in to the casting chamber. After polymerized of the resolving
gel, 4% of stacking gel will be transferred in to the gel plates(on top of running gel and then
appropriate comb will be inserted and allowed the stacking gel to solidified for 1hr at room
temperature and then carefully removed the comb (Davis and Goldberg, 2012).
The the LPSs sample wil be mixed with 5x sample buffer at equal amount and it wil be
keptin boiling water bath for 5 min before loading the gel (Kalambhe et al., 2017). Then
about 10 µl of the LPS will be loaded in a well. The gel will be run at 80V till (stacking gel)
for 15 minutes and further at 150 V (in separating gel) for 45 miniutes. After completion, the
gel will be removed from the apparatus and placed into a small tray and the SDS-PAGE gels
will be placed in Coomassie brilliant blue (0.25 %) staining solution for 2hrs and then
transferred to the de-staining solution for 8 hrs with frequent changes of de-staining solution.
Finally the result will be observed (Rezania et al., 2011).

38
4.4. Expermental Animal Selection and Management (Ethical Based)

This research study primarily involves experimental mice animal; both the contents and
conduct of the work do not in any way transgress the rights of experimental animals. For
immunization purpose pathogen free 40 female mouse (6 week aged, body weight from 21 to
23 gm) will be selected. The selected mouse will be acclimation for 7 days befor immunized
and all the necessary safety measures will be taken during the selection, acclimation,
handling and housing activity.
Fortunately the supervisor of this work is a veterinarian with extensive experience of
handling experimental animals. During injection as well as serum sample collection sterile
needles and syringes of appropriate gauge will be used.
And also animals will handled as per the international guiding principles for biomedical
research developed by the Council for International Organization of Medical Sciences
(CIOMS) and in accordance with local laws and regulations. Study animals will also be
handled and treated humanely and infected animals will be treated with appropriate drugs.
Animal will be provided with feed and water, their bedding materials will be changed
regularly and the general health of the animals will be monitored.

4.5. Production and Purification of Mice Polyclonal Antibodies Against STEC Strain

Immunization for routine polyclonal antibody production will be administered within 2 to 3

week interval until the desired amount of antibody is elicited. In this study pathogen free 40
female mice (6 week aged, body weight from 21 to 23 gram) for experiment and control will
purchased from Ehiopia Puplic Health institute. These mice will be acclimatized for 7 days
and keep them at sterilized condition.
Before starting immunization near to 0.05 ml of pre-immune control serum will be collected
from each mice by bleeding the ear of mices . This serum will be used as a negative control
and after that the injection site of mouse will be rinsed to remove debris and disinfected with
70% ethanol. The required amount of generated LPS will be mixed with equal amount of 2%
alum (AlHO3) adjuvant prior to immunization. Then on day 0 each experimental mices will
immunized with 50 μl of emulsion (25 μl LPS with 25 μl alum adjuvant) intrapereitoneally
(IP) by using ≤ 25G needle as a 1st immunization, after 2 weeks (day 14), interval of 1 st

39
immunized bleed (firs immune response) will collected from each mice. Then after two
weeks about 25 μl of emulsion (12.5 μg LPS antigen and 25 μl alum adjuvant) will be
injected in to each mouse intrapereitoneally as 1st boosting immunization. Following the 2nd
injection after 1 week (day 21) of 1 st boosting immunization, bleeds will be collected from
each mice through the tail vein by using 3ml syringe needles and then the level of
immunization response (anti-antigen activity) will be evaluated by using slide agglutination
test. After 2 weeks (day 35) of the 1 st boosting immunization, each mices will be boosted
with 25 μg of LPS antigen as 2 nd boosting immunization. After 1 week of the 2 nd boosting
near to 0.02- 0.05 ml of serum will be collected from each mice through the tail vein and at
2 week interval from the 2nd boost each mices will be boosted with 25 μg of LPS antigen as
3rd boosting immunization. One week after the 3rd boosting immunizations around the whole
blood (0.5 ml) will be collected from each mices.
The collected pre-immunized serum, 1st bleeds, 2nd bleeds and 3rd bleeds from each mices will
be pooled as control blood, blood of 1 st immunized mices (primery immune response), blood
of 1st boosted mices(secondary immune response), blood of 2 nd boosted mices(secondary
immune response) respectivelly and these collected blood will be keept at room temperature
for 30 minits and then after overnight waited the bleeds will be centrifuged at 4000 rpm for
10 minutes, the serum part will be separated from the whole blood and transferred into 2 ml
eppendorf tubes using a pipette. The separated serum thus obtained will be control serum,
primery immune response polyclonal antibody serum and secondary immune response
polyclonal antibody serum and stored at -20 0C for further experimentation (diagnostic assay
kit development) (He et al.,2013; Leenaars et al, 2005). For any step, the mouse will be
observed daily for a minimum of 15 minutes post-injection for any responses at the injection
sites in particular and for overall health or distress in general. At each step name of
immunogen, adjuvant used, route of administration, site(s) and volume of injection and date
of injection will be recorded.

40
4.6. Development of slide agglutination immunodiagnostic assay for STEC strain
detection using LPS and colony of STEC strains

In this experiment to develop or designied immunodiagnostic slide agglutination assay for

STEC strain detection using LPS of STEC strains, microscopy slide will be prepared with
appropriate labelled and circle them in to three parts with marking pen. About one drop (20
µL) of serum polyclonal antibody against LPS of STEC strain will be added in to separate
circles on a glss microscopy slide.

Then 1 drop (20 µL) of LPS (antigen) of STEC will be added in to each circle and carefully
mixed each solution with a separated side of the applicator stick and spread over the entire
area enclosed by the circle. Make sure the entire surface of the bottom of the circle is covered
with the mixture and falls complete to the end of the slide. Then the slide will be tilted
(shacked) slowly by rotated manually. Then the agglutination result will be observed within 1
to 2 minutes and recorded the results as POS, if agglutination occurs and scored on a scale
from + (barely visible) to +++( strong agglutination) or NEG, if there is no agglutination.

4.7. Evaluation of the specificity of the newly designed immunodiagnostic assay

To evaluate the specificity of the developed diagnostic assay in this study, the bacterial
strains such as 10 STEC, 9 EPEC, 3 EHEC, and 6 non – pathogenic strains and 4 non E. Coli
will be obtained from AAU, Institute of Biotechnology in the laboratory of the adviser as
glycerol stocks. The collected 10 STEC, 9 EPEC, 3 EHEC isolate will be cultured on EMB
medium and 4 non-Ecoli will be cultured on macConkey agar medium and incubated
aerobically overnight at 37 o C. the the specificity of the developed detection assay will be
checked wether detect STEC strain only or not by following the procedure described in
material and methods 4.6. Finally the specificity of the designed immunodiagnostic assay in
this study will be calculated in percent by using the formula of TN/ (TN + FP) × 100%;
where TN- represent true negative and FP- represent false positive data.

41
4.8. Evaluation of the sensitivity of the newly designed immunodiagnostic assay kit

To evaluate the sensetivity of the developed diagnostic assay in this study, Overnight cultures
of STEC will be serially diluted 10-fold. An aliquot of 20 𝜇L of the dilution will be plated on
the agar for viable count while another 20 𝜇L will be used for detection procedures of the
designed assays. Finally the sensitivity (capable of detecting as low as a single live bacterial
cell), of the designed immunodiagnostic assay in this study will be calculated in percent by
using the formula of (TP/ (TP + FN)) × 100%; where TP- represent true positive and FP-
represent false negative data.

4.9. Evaluating the detection capacity of the designed assays in human feace spiked with
STEC strain

To determine the detection capacity of the developed slide diagnostic assay in human face
spiked with STEC strain will be checked as followed: F eace will be obtained from a healthy
donor and processed immediately. Each stool sample (0.5 g) will be inoculated with 1 ml of 10-fold
serially diluted STEC strain overnight culture. From each dilution factor around 20 𝜇L of the
dilution will be plated on the agar for viable count while another 20 𝜇L will be used for
detection procedures of the designed assays. Finaly the detection capacity of the developed
assay kit will be decided on spike sample.

42
 5. EXPECTED OUT PUTS

Upon successful completion of this research work;

 A polyclonal antibody specific to STEC diarrheagenic strains will be produced

 Immunodiagnostic assays (agglutination based) using polyclonal antibodies will be
designed for detection of STEC strain
 The sensitivity and specificity of the designed assays will be determined
 The performance of the designed assays in detection of STEC strain from spike samples
will be validated

6. DATA MANAGEMENT AND ANALYSIS

In this study the generated data or result as True Positive (TP) , True Negative (TN), False
Positive (FP) and False Negative (FN) will be entered into a computer. Then, the statistical
analysis will be analyzed using MedCalc Version 18.11.3 Software packages and the
senestivity, specificity, Positive predicted value, Negative predicted value and Accuracy of
the developed immunodiagnostic assay kit will be determined

43
 7. TIME LINE/WORK PLANE OF THE STUDY
This study will be started and completed based on the following time table that means from
October 2017 to Jun 2019.
Activities 2017 2018 2019
Nov Dec Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr
Proposal preparation
and defense
Purchasing and
collection of
chemicals, reagents,
apparatus and
stationary materials
and arrangement of the
laboratory.
1st Activity:collection
and culturing of E.coli
sample and
preparation of LPS of
STEC strain
2nd Activity:
Polyclonal antibody
specific to STEC
strain will be produced
and by using this
produced pAb ,
immunodiagnostic
assay kit will be
developed for STEC
detection
3rd Activity: the
sepecificity,
senesetivity of the
developed
immunodiagnostic
assay kit to detect
STEC strain will be
checked
4th Activity:
Detection of STEC
pathogenic E.coli from
spike sample by using
newly designed
immunodiagnostic
assays
Data management and
analysis
First draft thesis will
be Written and

44
submitted to advisor
Final thesis
submission and
defense

8. COST OF THE PROJECT

To conduct this research budget with a total amount of 296,930 ETB is required. The
budget will be used for the purchase of all needed chemicals, reagents, apparatus,
stationary materials, advisor’s supervision fee, training cost for statistical software, costs of
transportation, data collection, communication and other miscellaneous activities. Detail
description of the budget is listed below in the table.

Item Unit Quantity Unit price Total price in

in Birr Birr

Laboratory mice, feeds, management 20,000

Freund's Complete Adjuvant Ml 50 100 5,000

Freund's Incomplete Adjuvant Ml 50 100 5,000

3-ml glass syringes with 19-, 21-, and Box/ 3 100 3000
22-G needles 12pcs

10 mM PBS, pH 7.4, containing 3% Ml 500 - 2000

BSA

glycine saline buffer Ml 500 - 1000

p-nitrohenyl phosphate – pNPP/ G 500 - 2500

Tetramethylbenzidine-TMB

Horseradish Peroxidase- rabit anti- Pcs 5 2550 12,750

45
mouse conjugated enzyme lebeled
pAb, 200µg/0.5ml

Sodium azide G 50 1500 1,500

Clear Centrifuge eppendorf tubes, 5ml Pcs 1000 - 800

mCCDA (modified charcoal sephoprozon G 500 - 2100

deoxygalase agar

0.1 M glycine-HCl, pH 2.7. Ml 50 - 1000

BCA Protein Assay Kit Pcs 1 3000 3000

Acepromazine tablet Pcs 50 300 1500

Butorphanol, 2.5 ml of 10mg/ml Pcs 1 1200 1200

EMLA cream, 30 gram G 500 2000

60 tablet of Glycopyrrolate 1 mg Pcs 1 750 750

100mg/ml 50 ml Xylazine Pcs 1 1200 1200

100mg/ml 50 ml Ketamine Pcs 1 1200 1200

100mg/ml 50 ml Chlorhexidine Pcs 1 1200 1200

Saturated ammonium sulfate G 500 1000

Phosphate buffered saline (PBS) Ml 1000 - 2500

Multichannel micropipette (0.2- Pcs 5 700 3500

1000µl)

Microcentrifuge tubes (0.5 to 2.0 mL) Pcs 1500

46
Modified Buffered Peptone water with Ml 2000 - 2,000
pyruvate (mBPWp)

Water bath or heat block capable of Pcs 1 1000 1000

maintaining 100°C.

Disposable micropipette tips 0.2 to Pack 3 300 900

1000 µl (aerosol resistant tips)

Deionized or distilled Water, L 10 320 3200

Swap/stick with cotton Pack 4 50 200

Lab pipette round stand Pcs 1 1500 1500

Bursen burner Pcs 2 100 200

Inoculation needle Pcs 4 100 400

0.5 M NaOH (hydrolysis stopped) L 0.5 200 100

Glycerol L 1 100 100

Washing buffer L 0.5 1000 500

Flask Pcs 10 60 600

Aluminum foil Pcs 10 60 600

Eppindorf Pack 10 150 1500

Luria Bertani agar G 500 1200 1200

Glasswares Pcs 20 60 1200

Eosin Methylene Blue Agar G 500 1500 1500

Disposable latex gloves Pack 20 150 2400

47
Lab coat PCS 2 250 500

Eye glass Pcs 2 40 80

Safety mask (for nose) PCS 10 30 300

Erlenmeyer flask Pcs 4 100 400

Tris buffer L 1 1000 1000

Phosphate buffered saline Tablet 50tab 200 1000

Ethanol L 4 200 800

Inoculation loops Pcs 5 60 300

Sample cups
Pcs 50 40 2000
Ice box
Pcs 1 450 450
Measuring cylinder
Pcs 5 60 300
Spatula
Pcs 5 30 150
Liquid nitrogen cylinder
Pcs 1 12000 12,000
Premier EHEC commercial kit or
Pcs 1 14,000
ProSpecSTECMicroplate Assay
Incubators: 36°C ± 1°C and 42°C ±
Pcs 1 - 15,000
1°C
Autoclave
Pcs 1 - 15,000
Refrigerator
Pcs 1 10,000 10,000

Microtiter plate reader (optional) PCS 1 - -

spectrophotometer with 405-nm filter

48
Stablized 96- well microtiter plates PCS 1 10,000 10,000
Specimen storage vials
Pcs 25 30 1500
Petri dishes D90mm Pcs 25 30 750

D60mm Pcs 25 30 750

Test tubes
Pcs 50 30 1500
Thermometer Pcs 2 150.80 301.60

Droper Pcs 10 26 260

Test tube rack Pcs 2 650 1300

Sterile glass jars for enrichment Pcs 5 - 1500

Tryptone
G 500 1200 1200
Glycerol
L 1 250 250
Bovine serum albumin
G 10 2100 2100
Mac Conkey agar
G 500 1300 2600
MgCl2.
G 500 - 1400
CaCl2
G 500 1400
proteinase K
Ml 10 1500
RNase
Ml 10 1500
DNase
Ml 10 1500
MgSO4
G 500 500 500
Chloroform
Ml 500 500 500

49
90% phenol
Ml 500 1000 1000
0.5 M sodium acetate
Ml 10 1400 1400
silver and coomassie blue staining
Ml 10 1600 1600
Agarose 1%
G 50 2000 2000
Ethidium bromide (EtBr)
Ml 10 1000 1000
Electrophoresis buffer: 1% TBE
Ml 500 350 350
DNA loading dye
Pack 1 1900 1900

Ladder (DNA marker)

Pack 2 7000 14000
Nuclease-free H2O
Pack 2 180 360
DNTPs
Pack 2 5000 10,000
forward and reverse primer
Vial 11 1000 11000
Taq DNA Polymerase (5u/L)
Pack 1 3800 3800
Transportation and pere dime cost for
- - - 3600
materials purchas and data collection
advisor’s supervision fee
- - 3000
stationary materials & print 1000 1000

Total 258,200
00
Contingency (15%) 38,730

Grand total 296,930

50
 9. REFERENCE

Abdul Jabbar, and Richard A. (2003). Gastroenteritis and antibiotic-associated diarrhea.

Division of Gastroenterology/Hepatology, Department of Medicine, University of

51